TABLE OF CONTENTS
PREFACE—COURSE DESCRIPTION9
CHAPTER 1--MICROSCOPY 13
LIGHT MICROSCOPY .13
ELECTRON MICROSCOPY.16
MICROSCOPY APPENDIX. ALDEHYDE FIXATIVES 20
CHAPTER 2--SPECTROPHOTOMETRY23
SPECTROPHOTOMETRY THEORY 23
INSTRUMENTATION25
APPLICATIONS OF SPECTROPHOTOMETRY25
FACTORS AFFECTING ABSORPTION .26
VARIATIONS IN SPECTROPHOTOMETRY.26
APPENDIX I. PHOTOMULTIPLIER TUBE28
APPENDIX II. CALCULATION OF ENZYME ACTIVITY.29
CHAPTER 3--FLUORESCENCE33
ENZYME ASSAYS AND FLUOROMETERS.33
FLUORESCENCE MICROSCOPY.34
FLOW CYTOMETRY .37
CHAPTER 4--RADIOCHEMISTRY .39
ATOMIC STRUCTURE AND RADIOACTIVE DECAY39
PROPERTIES OF RADIOISOTOPES 40
MEASUREMENT OF ß-RADIATION .41
GAMMA-RAY DETECTION .46
SAFETY .46
APPENDIX 1. INTERNAL STANDARDS.47
APPENDIX 2. CHANNELS RATIO .48
APPENDIX 3. MULTIPLE ISOTOPES 49
CHAPTER 5--pH AND BUFFERS51
DEFINITIONS .51
HENDERSON-HASSELBACH.51
INSTRUMENTATION52
COMPLICATIONS52
BUFFERING CAPACITY .53
BUFFER SELECTION 53
BUFFER APPENDIX 54
CHAPTER 6--CENTRIFUGATION57
SEDIMENTATION THEORY.57
PREPARATIVE CENTRIFUGATION59
CENTRIFUGATION APPENDIX 1. RCF CALCULATION.62
CHAPTER 7--INTRODUCTION TO PROTEINS65
PROTEIN STRUCTURE .65
PROTEIN STABILITY67
PROTEIN ASSAYS .68
APPENDIX. AMINO ACIDS: PROPERTIES AND STRUCTURE.70
CHAPTER 8--DIFFERENTIAL PRECIPITATION OF PROTEINS 73
PROCEDURE FOR (NH4)2SO4 PRECIPITATION.73
OTHER PROTEIN PRECIPITATION METHODS 74
APPENDIX. AMMONIUM SULFATE NOMOGRAM75
CHAPTER 9--CHROMATOGRAPHY .77
BASIC PRINCIPALS.77
EQUIPMENT .78
ADSORPTION CHROMATOGRAPHY.79
ION EXCHANGE CHROMATOGRAPHY 79
HYDROPHOBIC CHROMATOGRAPHY .83
Polar Solvent84
GEL FILTRATION CHROMATOGRAPHY84
AFFINITY CHROMATOGRAPHY85
CHAPTER 10--MEMBRANES AND DETERGENTS.87
TYPES OF MEMBRANE PROTEINS88
DETERGENTS.89
DETERGENT REMOVAL 91
APPENDIX 1. CELL DISRUPTION TECHNIQUES.92
CHAPTER 11--ELECTROPHORESIS95
GEL ELECTROPHORESIS.95
EQUIPMENT .97
SDS-PAGE .98
ISOELECTRIC FOCUSING100
TWO-DIMENSIONAL GEL ELECTROPHORESIS101
PROTEIN DETECTION FOLLOWING ELECTROPHORESIS102
PREPARATIVE ELECTROPHORESIS105
ELECTROPHORESIS APPENDIX 1. SIZE CALCULATION. .108
ELECTROPHORESIS APPENDIX 2. GEL STAINING 109
CHAPTER 12--PROTEIN PURIFICATION OVERVIEW.111
MICROSEQUENCING AND PEPTIDE MAPPING 114
APPENDIX. FOLD PURIFICATION AND RECOVERY .117
CHAPTER 13--IMMUNIZATION121
IMMUNOGENICITY 121
IMMUNOGEN PREPARATION.122
IMMUNIZATION123
COLLECTING AND PROCESSING BLOOD .126
ANTIBODY PURIFICATION.126
CHAPTER 14--MONOCLONAL ANTIBODIES.129
IMMUNIZATION129
FUSION130
SELECTION.130
SCREENING131
CLONING 131
PRODUCTION.132
APPENDIX. B-CELL DIFFERENTIATION 133
CHAPTER 15--IMMUNOASSAYS.134
IMMUNOPRECIPITATION .135
IMMUNOBLOTTING .137
IMMUNOFLUORESCENCE 139
IMMUNOGOLD ELECTRON MICROSCOPY .140
ELISA (AND RIA).141
DIRECT VS. INDIRECT .142
OTHER METHODS.144
CHAPTER 16--NUCLEIC ACID STRUCTURE AND ISOLATION.149
ISOLATION OF NUCLEIC ACIDS150
ISOLATION OF RNA .153
DENSITY GRADIENT CENTRIFUGATION154
ANALYSIS AND QUANTIFICATION154
APPENDIX 1. COMMON CONVERSIONS 155
CHAPTER 17--MODIFICATION OF NUCLEIC ACIDS 157
RESTRICTION ENDONUCLEASES .157
CHAPTER 18--ELECTROPHORESIS OF NUCLEIC ACIDS .161
PULSE FIELD GEL ELECTROPHORESIS .163
CHAPTER 19--HYBRIDIZATION AND BLOTTING TECHNIQUES165
GENERAL PROCEDURES.165
FACTORS AFFECTING HYBRIDIZATION.167
PREPARATION OF LABELED DNA PROBES168
RFLP AND RESTRICTION MAPPING .171
IN SITU HYBRIDIZATION172
DNA MICROARRAYS173
CHAPTER 20--POLYMERASE CHAIN REACTION175
PCR MECHANISM175
PRACTICAL ASPECTS 176
RNA-PCR .178
QUANTITATIVE PCR 178
LIMITATIONS.180
PRECAUTIONS.182
CHAPTER 21--RECOMBINANT DNA 183
PREPARATION OF FOREIGN DNA.183
PLASMID VECTORS186
BACTERIOPHAGE λ 192
FILAMENTOUS BACTERIOPHAGE196
COSMIDS AND YACS .198
QUALITY CONTROL.199
CHAPTER 22--EXPRESSION OF RECOMBINANT PROTEINS.201
EXPRESSION IN E. COLI.201
PREPARATION OF EXPRESSION VECTORS.203
SCREENING λ LIBRARIES WITH ANTIBODIES.203
EXPRESSION IN EUKARYOTES204
APPENDIX 1. CODES AND CODONS206
APPENDIX 2. FUSION PROTEINS .207
APPENDIX 3. GENERATING FRAMESHIFTS208
CHAPTER 23--DNA SEQUENCING 209
DIDEOXY CHAIN TERMINATION209
EXTENDED SEQUENCING STRATEGIES212
MAXIM AND GILBERT.215
CHAPTER 24--SEQUENCE ANALYSIS AND BIOINFORMATICS 216
SEQUENCE ALIGNMENT.216
SEARCHING DATABASES.218
PROTEOMICS.219
APPENDIX 1. WEBSITES.224
APPENDIX 2. RESULTS OF BLAST SEARCH.225